The primary objective of this mentored clinical scientist award is to acquire the skills and expertise to become an independent investigator in the rheumatic diseases. This will occur through a period of intensive didactic and research training in the Division of Rheumatology at the Johns Hopkins University School of Medicine. The scientific objective of this application is to define mechanisms of autoantigen selection in systemic autoimmunity, a process that remains largely unknown. We propose that (i) frequently targeted autoantigens stimulate toll-like receptors (TLRs), that (ii) antigens acquire this capacity through posttranslational modification, and that (iii) posttranslationally modified antigens may induce systemic auto-immunity. The DNA repair enzyme Poly(ADP-ribose) polymerase 1 (PARP-1) is a frequent target in human SLE and a universal target in the parent-into-F1 (BDF1) mouse model of SLE. Upon activation, PARP-1 modifies itself by poly(ADP-ribosyl)ation and our preliminary data in mouse macrophages suggests that this modification enables PARP-1 to activate TLRs. In this application we will (i) define the specific TLR target of poly(ADPribosyl)ated PARP-1. We will stimulate mouse macrophages deficient in individual TLRs with poly(ADPribosyljated PARP-1. Selective abrogation of TLR effector pathway activation will indicate the target TLR. For further confirmation the target TLR will be transfected into TLR-devoid HEK293 cells along with an NFkappa B driven luciferase construct and stimulated with poly(ADP-ribosyl)ated PARP-1. By injection of poly(ADP-ribosyl)ated PARP-1 into wild-type or TLR-deficient mice we will assess TLR-dependent activation of splenocytes in vivo, (ii) We will define whether activation of the poly(ADP-ribose) pathway is required for the evolution of PARP-1 into an autoantigen in vivo. We will administer inhibitors of the poly(ADP-ribose) pathway during disease induction in the BDF1 model of SLE and compare anti-PARP-1 antibody liters between disease and control animals, (iii) We will determine the role of TLR activation for the induction of the antibody response to PARP-1 in vivo. Anti-PARP-1 antibody titers in BDF1 mice deficient in MyD88 (a critical TLR pathway adaptor molecule) will be compared to BDF1 wild-type mice after disease induction. The insights provided by determining the role of poly(ADP-ribosyl)ation for the capacity of PARP-1 to stimulate TLRs and induce anti-PARP-1 antibodies in SLE will likely prove to be relevant for defining mechanisms for the reversible acquisition of TLR-ligand activity by other autoantigens, and thus may reveal novel mechanisms and therapy for other systemic autoimmune diseases. Relevance: This study defines mechanisms for disease induction in systemic autoimmunity. It investigates the hypothesis that autoantigens themselves play an active role for the shaping of the immune response through activation of the innate immune system. The insights from this study may lead to novel therapies in systemic autoimmunity. [unreadable] [unreadable] [unreadable] [unreadable]